IL-17, IL-6, IL-1β, and TNFα cytokine concentrations were determined in plasma samples using commercially available human ELISA kit (human IL-17, DY317-05; human IL-6, DY206-05; human IL-1β, DY201-05 [R&D systems, MN, USA]; and human TNFα, ab181421; Abcam, Cambridge, MA, USA). Assays were preformed following the manufacturer's instructions. All samples were measured in duplicates.
Human il 17
Manufactured by R&D Systems
Sourced in United States
Human IL-17 is a recombinant human protein that functions as a proinflammatory cytokine. It is involved in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ab181421, by Abcam (1 mentions)
Human il 6, by R&D Systems (1 mentions)
Sunrise absorbance reader, by Tecan (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Anti cd3 mab coated beads, by Thermo Fisher Scientific (1 mentions)
Aeb071, by Novartis (1 mentions)
Human il 1β, by R&D Systems (1 mentions)
Human tnf α elisa kit, by Abcam (1 mentions)
Dy317 05, by R&D Systems (1 mentions)
Human gm csf, by R&D Systems (1 mentions)
Human ifn γ, by R&D Systems (1 mentions)
Iscove modified dulbecco medium, by Thermo Fisher Scientific (1 mentions)
Il 17, by R&D Systems (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
6 protocols using human il 17
1
Quantifying Inflammatory Cytokines
2
ELISA-based IL-10 and IL-17 Quantification
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IL-10 and IL-17 concentrations were measured using ELISA kits for human IL-10 (BD Biosciences) or human IL-17 (R&D Systems, Minneapolis, MN, USA) according to the manufacturers’ instructions. Levels were estimated by interpolation from a standard curve generated using a Sunrise absorbance reader (Tecan, Männedorf, Switzerland) at 450 nm.
3
Tregs Suppress T Cell Proliferation
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Different subsets of Tregs were treated or not, washed three times, and added at ratio 1:3 (1.25 × 105 Tregs: 5 × 105 CD4+CD25− T cells) to CD4+CD25− T cells at final concentration 2 × 106/ml. The cells were co-cultured on anti-CD3 mAb (5 μg/ml) pre-coated 24-well plates for 48 hr. The PKC-θ inhibitor, AEB071, was provided by Novartis (Ridgefield, CT) and dissolved in DMSO. T cells were pretreated for 30 min with 10 μM at 37 °C and washed three times. Cytokine secretion was determined by ELISA as previously described20 (link), using Human IL-17 (R&D Systems Inc, Minneapolis, MN) and IFN-γ Cytosettm (Biosource; Camarillo, CA). The capacity of expanded Tregs to suppress target cell proliferation in vitro was assessed by using 5-carboxyfluorescein-diacetate-succinimide ester (CFSE) inhibition assay31 (link). Peripheral blood mononuclear cells (PBMCs) were purified, labeled with CFSE (InVitrogen), and stimulated with anti-CD3 mAb-coated beads (Dynal) in 96-well round-bottom plates with or without expanded cells (Treg:PBMC at ratios of 1:4-1:64). Assays were harvested on day 4. Cells were stained with anti-CD4 and/or −8 mAb and data acquired by LSRII. Data was analyzed using the proliferation platform in FlowJo (Treestar, Ashland, OR), and suppression determined from the Division Index.
4
Measuring Salivary Cytokine Levels
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IL-17, IL-1β and TNFα cytokine concentrations were determined in saliva and/or plasma samples using commercially available human ELISA kits (Human IL-17, DY317-05, R&D; Human IL-1β, DY201-05, R&D; and human TNFα ELISA KIT, ab181421, Abcam). For the assays, saliva samples were centrifuged at 700g for 15 minutes at 4°C, and the supernatant was used. Diluent optimization was performed for the saliva samples. We performed assays following the manufacturers’ instructions. All samples were measured in duplicates.
5
Cytokine Profiling of Activated Murine and Human Immune Cells
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Spleen cells were cultured in Iscove Modified Dulbecco medium (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gibco), 5% l -glutamine (Gibco), 5% penicillin/streptomycin (Gibco), and β-mercaptoethanol (Sigma). Spleen cells were stimulated with 1 μg/mL anti-CD3/CD28 agonistic antibodies for 72 hours at 37°C, 5% CO2. For antigen-specific recall response, CNS-infiltrating cells from EAE mice, at a concentration of 1 × 106 cells/mL, were stimulated with 10 μg/mL MOG35–55 at 37°C, 5% CO2. At the indicated time points, supernatants were collected and centrifuged to eliminate cellular debris. Cytokine levels in supernatants were measured by ELISA. We analyzed the following cytokines: GM-CSF (R&D Systems, Minneapolis, MN, DY415), human GM-CSF (R&D Systems DY215), IFN-γ (R&D Systems DY485), human IFN-γ (R&D Systems DY285), IL-17 (R&D Systems DY421), and human IL-17 (R&D Systems DY317).
6
Cytokine Stimulation of Cell Lines
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Culture conditions of primary cells are described in the Supplementary Materials and Methods. A HaCaT human epidermal keratinocyte cell line (Boukamp et al., 1988 (Boukamp et al., , 1997) ) was obtained from ScienCell Research Laboratories (Carlsbad, CA). An H4 human cancer cell line was purchased from ATCC (Sumitomo Dainippon Pharma, Osaka, Japan). All cell lines were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA) with 10% fetal bovine serum (Thermo Fisher Scientific) without antibiotics at 37 C under 5% CO 2 . For cytokine stimulation, cells were plated in 12-well plates (1 Â 10 5 cells/well) and stimulated with human IL-6 (100 ng/ml; Toray Industries, Tokyo, Japan) plus human soluble IL-6 receptor (100 ng/ml; R&D Systems, Minneapolis, MN) and/or human IL-17 (100 ng/ml; R&D Systems) as well as TNF-a (50 ng/ml; PeproTech, Tokyo, Japan) for 1 or 3 hours after 2 hours of serum starvation. After stimulation, the cells were harvested, and total RNA was prepared for real-time PCR.
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